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Despite being commonplace, polymerase chain reaction (PCR) still contains many unknown aspects. One example is microsatellite PCR, which is now widely used for various purposes from ecology to cancer medicine. Since this category of repetitive DNA sequences induces polymerase slippage not only in vivo but also in vitro, microsatellite PCR products comprise a complex combination of DNA fragments with various lengths and have, therefore, been empirically interpreted. The primary obstacle for understanding microsatellite PCR was the intrinsic inaccuracy in sizing of DNA fragments in capillary electrophoresis, which, however, has been overcome by elucidating intrinsic sizing errors in each fragment length range. Secondly, the slippage properties of the thermostable polymerases were first clarified in detail using primer extension assays. Furthermore, using the obtained slippage parameters and our original program, we have first reconstructed microsatellite PCR in silico. The entire processes of complex microsatellite PCR have thus been more clearly understood. This article is protected by copyright. All rights reserved.

To implement precision oncology, analytical validity as well as clinical validity and utility are important. However, proficiency testing (PT) to assess validity has not yet been systematically performed in Japan. To investigate the quality of next-generation sequencing (NGS) platforms and cancer genome testing prevalent in laboratories, we performed pilot PT using patient samples. We prepared 5 samples from patients with lung or colorectal cancer, extracted genomic DNA from the cancer tissue and peripheral blood, and distributed these to 15 laboratories. Most participating laboratories successfully identified the pathogenic variants, except for two closely located KRAS variants, and 25-bp delins in the EGFR exon 19, not identified by the in vitro diagnostics testing. Conversely, the EGFR L858R variant was successfully identified, and the allele frequency was similar for all the laboratories. A high DNA integrity number led to excellent depth and reliable NGS results. All NGS platforms and bioinformatics pipelines have advantages and disadvantages. We propose the use of a PT program using patient samples to ascertain the quality status of cancer gene testing in laboratories and to ensure that laboratories have sufficient information to develop advancements in precision medicine for cancer.

Titanium-n-butoxide was hydrolyzed in the presence of benzoylacetone, and the resulting solution was concentrated and dried at 120 or 140 °C to obtain transparent amorphous materials. High-energy X-ray diffraction measurement was conducted at the SPring-8 facility, and the reduced pair distribution function, G(r) was calculated by Fourier transform of the total structure factor, S(Q). The G(r) value suggested that the materials are composed of TiO6 octahedra linked by corner- and edge-sharing. Low temperature thermomechanical analysis (TMA) and differential scanning calorimetry (DSC) were conduced on the materials, where a deflection was detected both in the TMA and DSC curves, revealing the glass transition of the materials. Combined with the previous work based on infrared absorption spectroscopy and gel permeation chromatography, the materials are demonstrated to be a new class of glassy materials composed of linked metal-oxygen polyhedra chelated with organic molecules. The materials are innovative due to the high refractive indices that originate in the metal-oxo oligomers and to the shapability given by their thermoplastic properties.

DNA mismatch repair (MMR) is an evolutionally conserved and major mechanism in cells that counteracts the occurrence of point mutations on the genome. Defective MMR confers the ‘mutator’ phenotype ...

In prokaryotes and yeasts, DNA polymerase proofreading (PPR) and DNA mismatch repair (MMR) cooperatively counteracts replication errors leading to repeat sequence destabilization (i.e. insertions/deletions of repeat units). However, PPR has not thus far been regarded as a mechanism stabilizing repeat sequences in higher eukaryotic cells. In a human cancer cell line, DLD-1, which carries mutations in both MSH6 and the Exo domain of POLD1, we previously observed that mononucleotide microsatellites were markedly destabilized whereas being stable in the simple MMR-defective backgrounds. In this study, we introduced the Exo domain mutation found in DLD-1 cells into MSH2-null HeLa cell clones, using CRISPR/Cas9 system. In the established Exo-/MMR-mutated HeLa clones, mononucleotide repeat sequences were remarkably destabilized as in DLD-1 cells. In contrast, dinucleotide microsatellites were readily destabilized in the parental MMR-deficient backgrounds, and the instability was not notably increased in the genome-edited HeLa clones. Here, we show an involvement of the Exo domain functions of DNA polymerase delta in mononucleotide repeat stabilization in human cells, which also suggests a possible role division between DNA polymerase and MMR in repeat maintenance in the human genome.

Human papillomavirus (HPV)-related oropharyngeal small-cell carcinoma (OPSmCC) is a rare malignancy with aggressive behavior, whereas HPV-related oropharyngeal squamous-cell carcinoma (OPSqCC) displays a favorable prognosis. Notably, these two malignancies occasionally arise in an identical tumor. In this case study, we explored the molecular characteristics that distinguishes these two carcinomas using a rare case of HPV-related oropharyngeal carcinoma (OPC) with the combined histology of SmCC and SqCC. Immunohistochemical analysis and HPV-RNA in situ hybridization (ISH) suggested that both SmCC and SqCC were HPV-related malignancies. Targeted exome sequencing revealed that SmCC and SqCC had no significant difference in mutations of known driver genes. In contrast, RNA sequencing followed by bioinformatic analyses suggested that aberrant transcriptional programs may be responsible for the neuroendocrine differentiation of HPV-related OPC. Compared to SqCC, genes up-regulated in SmCC were functionally enriched in inflammatory and immune responses (e.g., arachidonic acid metabolism). We then developed a SmCC-like gene module (top 10 up-regulated genes) and found that OPC patients with high module activity showed poor prognosis in The Cancer Genome Atlas (TCGA) and GSE65858 cohort. Gene set enrichment analysis of the SmCC-like gene module suggested its link to MYC proto-oncogene in the TCGA data set. Taken together, these findings suggest that the SmCC-like gene module may contribute to acquisition of aggressive phenotypes and tumor heterogeneity of HPV-related OPC. The present case study is the first report of genetic and transcriptomic aberrations in HPV-related OPSmCC combined with SqCC.

This study examined the potential pathogenicity of Shiga toxin-producing Escherichia coli (STEC) in feces of sika deer by PCR binary typing (P-BIT), using 24 selected STEC genes. A total of 31 STEC strains derived from sika deer in 6 prefectures of Japan were O-serotyped and found to be O93 (n=12), O146 (n=5), O176 (n=3), O130 (n=3), O5 (n=2), O7 (n=1), O96 (n=1), O116 (n=1), O141 (n=1), O157 (n=1) and O-untypable (n=1). Of the 31 STEC strains, 13 carried both stx1 and stx2, 5 carried only stx1, and 13 carried one or two variants of stx2. However, no Stx2 production was observed in 3 strains that carried only stx2: the other 28 strains produced the appropriate Stx. P-BIT analysis showed that the 5 O5 strains from two wild deer formed a cluster with human STEC strains, suggesting that the profiles of the presence of the 24 P-BIT genes in the deer strains were significantly similar to those in human strains. All of the other non-O157 STEC strains in this study were classified with strains from food, domestic animals and humans in another cluster. Good sanitary conditions should be used for deer meat processing to avoid STEC contamination, because STEC is prevalent in deer and deer may be a potential source of STEC causing human infections.

Purpose Microsatellite instability (MSI) has been a long-standing biomarker candidate for drug resistance in tumour cells. Despite numerous clinical studies, the data in the literature are not conclusive. The complexity of the MSI phenomenon in some malignancies may, at least partly, account for the discrepancy. In addition, methodological problems are also pointed out in the assay techniques. We previously established a unique fluorescent technique in which the major methodological problems in conventional assays are overcome. Application of this technique has revealed two distinct modes of microsatellite alterations, i.e. Type A and Type B. More importantly, we demonstrated that Type A MSI is the direct consequence of defective DNA mismatch repair (MMR) that causes cellular resistance against antineoplastic agents. Method We first applied this technique to adult T-cell leukaemia/lymphoma (ATLL). Results The MSI phenomenon was indeed observed in ATLLs (4/20, 20%). Intriguingly, the observed microsatellite alterations were invariably Type A, which implies that the tumours were MMR-defective. Indeed, clinical outcomes of patients with these MSI+ tumours were significantly worse. Furthermore, multivariate analysis revealed that Type A MSI is an independent prognostic factor. Conclusion These observations strongly suggest the possibility of Type A MSI as a prognostic and potentially predictive biomarker in ATLL.

To investigate the molecular mechanism(s) by which herpes simplex virus 1 (HSV-1) tegument protein UL51 promotes viral replication, we screened for viral proteins that interact with UL51 in infected cells. Affinity purification of tagged UL51 in HSV-1-infected Vero cells was coupled with immunoblotting of the purified UL51 complexes with various antibodies to HSV-1 virion proteins. Subsequent analyses revealed that UL51 interacted with another tegument protein, UL14, in infected cells. Mutational analyses of UL51 showed that UL51 amino acid residues Leu-111, Ile-119, and Tyr-123 were required for interaction with UL14 in HSV-1-infected cells. Alanine substitutions of these UL51 amino acid residues reduced viral replication and produced an accumulation of unenveloped and partially enveloped nucleocapsids in the cytoplasm at levels comparable to those of UL51-null, UL14-null, and UL51/UL14 double-null mutations. In addition, although UL51 and UL14 colocalized at juxtanuclear domains in HSV-1-infected cells, the amino acid substitutions in UL51 produced aberrant localization of UL51 and UL14. The effects of these substitutions on localization of UL51 and UL14 were similar to those of the UL51-null and UL14-null mutations on localization of UL14 and UL51, respectively. These results suggested that the interaction between UL51 and UL14 was required for proper localization of these viral proteins in infected cells and that the UL51-UL14 complex regulated final viral envelopment for efficient viral replication. IMPORTANCE Herpesviruses contain a unique virion structure designated the tegument, which is a protein layer between the nucleocapsid and the envelope. HSV-1 has dozens of viral proteins in the tegument, which are thought to facilitate viral envelopment by interacting with other virion components. However, although numerous interactions among virion proteins have been reported, data on how these interactions facilitate viral envelopment is limited. In this study, we have presented data showing that the interaction of HSV-1 tegument proteins UL51 and UL14 promoted viral final envelopment for efficient viral replication. In particular, prevention of this interaction induced aberrant accumulation of partially enveloped capsids in the cytoplasm, suggesting that the UL51-UL14 complex acted in the envelopment process but not in an upstream event, such as transport of capsids to the site for envelopment. This is the first report showing that an interaction between HSV-1 tegument proteins directly regulated final virion envelopment.

Noninvasive positive pressure ventilation (NPPV) using a helmet is expected to cause inspiratory trigger delay due to the large collapsible and compliant chamber. We compared the work of breathing (WOB) of NPPV using a helmet or a full face-mask with that of invasive ventilation by tracheal intubation. We used a lung model capable of simulating spontaneous breathing (LUNGOO; Air Water Inc., Japan). LUNGOO was set at compliance (C) = 50 mL/cmH2O and resistance (R) = 5 cmH2O/L/s for normal lung simulation, C = 20 mL/cmH2O and R = 5 cmH2O/L/s for restrictive lung, and C = 50 mL/cmH2O and R = 20 cmH2O/L/s for obstructive lung. Muscle pressure was fixed at 25 cmH2O and respiratory rate at 20 bpm. Pressure support ventilation and continuous positive airway pressure were performed with each interface placed on a dummy head made of reinforced plastic that was connected to LUNGOO. We tested the inspiratory WOB difference between the interfaces with various combinations of ventilator settings (positive end-expiratory pressure 5 cmH2O; pressure support 0, 5, and 10 cmH2O). In the normal lung and restrictive lung models, WOB decreased more with the face-mask than the helmet, especially when accompanied by the level of pressure support. In the obstructive lung model, WOB with the helmet decreased compared with the other two interfaces. In the mixed lung model, there were no significant differences in WOB between the three interfaces. NPPV using a helmet is more effective than the other interfaces for WOB in obstructive lung disease.

Herpes simplex virus 1 (HSV-1) UL51 is a phosphoprotein that functions in the final envelopment in the cytoplasm and viral cell-cell spread, leading to efficient viral replication in cell cultures. To clarify the mechanism by which UL51 is regulated in HSV-1-infected cells, we focused on the phosphorylation of UL51. Mass spectrometry analysis of purified UL51 identified five phosphorylation sites in UL51. Alanine replacement of one of the identified phosphorylation sites in UL51, serine 184 (Ser-184), but not the other identified phosphorylation sites, significantly reduced viral replication and cell-cell spread in HaCaT cells. This mutation induced membranous invaginations adjacent to the nuclear membrane, the accumulation of primary enveloped virions in the invaginations and perinuclear space, and mislocalized UL34 and UL31 in punctate structures at the nuclear membrane; however, it had no effect on final envelopment in the cytoplasm of HaCaT cells. Of note, the alanine mutation in UL51 Ser-184 significantly reduced the mortality of mice following ocular infection. Phosphomimetic mutation in UL51 Ser-184 partly restored the wild-type phenotype in cell cultures and in mice. Based on these results, we concluded that some UL51 functions are specifically regulated by phosphorylation at Ser-184 and that this regulation is critical for HSV-1 replication in cell cultures and pathogenicity in vivo. IMPORTANCE HSV-1 UL51 is conserved in all members of the Herpesviridae family. This viral protein is phosphorylated and functions in viral cell-cell spread and cytoplasmic virion maturation in HSV-1-infected cells. Although the downstream effects of HSV-1 UL51 have been clarified, there is a lack of information on how this viral protein is regulated as well as the significance of the phosphorylation of this protein in HSV-1-infected cells. In this study, we show that the phosphorylation of UL51 at Ser-184 promotes viral replication, cell-cell spread, and nuclear egress in cell cultures and viral pathogenicity in mice. This is the first report to identify the mechanism by which UL51 is regulated as well as the significance of UL51 phosphorylation in HSV-1 infection. Our study may provide insights into the regulatory mechanisms of other herpesviral UL51 homologs.

// Asuka Murata 1, * , Yoshifumi Baba 1, * , Takatsugu Ishimoto 1 , Keisuke Miyake 1 , Keisuke Kosumi 1 , Kazuto Harada 1 , Junji Kurashige 1 , Shiro Iwagami 1 , Yasuo Sakamoto 1 , Yuji Miyamoto 1 , Naoya Yoshida 1 , Manabu Yamamoto 2 , Shinya Oda 3 , Masayuki Watanabe 4 , Mitsuyoshi Nakao 5 , Hideo Baba 1 1 Department of Gastroenterological Surgery, Graduate School of Medical Science, Kumamoto University, Japan 2 Department of Surgery, National Hospital Organization Kyushu Cancer Center, Japan 3 Department of Cancer Biology, National Kyushu Cancer Center Clinical Research Institute, Japan 4 Department of Gastroenterological Surgery, Cancer Institute Hospital, Japanese Foundation for Cancer Research, Japan 5 Department of Medical Cell Biology, Institute of Molecular Embryology and Genetics, Kumamoto University, Japan * These authors have contributed equally to this work Correspondence to: Yoshifumi Baba, e-mail: y-baba@kumamoto-u.ac.jp Keywords: epigenetics, markers, demethylation, TET Received: April 17, 2015 Accepted: May 26, 2015 Published: June 08, 2015 ABSTRACT Mammalian DNA is epigenetically marked by 5′-cytosine methylation (5-methylcytosine [5-mC]). The Ten-eleven translocation (TET) enzymes (TET1, TET2, and TET3) are implicated in DNA demethylation, through dioxygenase activity that converts 5-mC to 5-hydroxymethylcytosine (5-hmC). Although decreased TET is reportedly associated with decreased 5-hmC levels in various cancers, functions of 5-hmC and TET expression in esophageal squamous cell carcinoma (ESCC) are unclear. We used ELISA and immunohistochemistry tests to analyze 5-hmC status in ESCC tissues, RT-qPCR to analyze TET family mRNA expression in normal and tumor tissues, and pyrosequencing to quantify LINE-1 (i.e., global DNA methylation) levels. ELISA and immunohistochemical testing showed 5-hmC levels were significantly lower in ESCC than in paired normal tissues ( P < 0.0001). TET2 expression was significantly lower in ESCCs than paired normal tissues ( P < 0.0001), and significantly associated with 5-hmC levels in ESCCs ( P = 0.003, r = 0.33). 5-hmC levels were also significantly associated with LINE-1 methylation level ( P = 0.0002, r = 0.39). Patients with low 5-hmC levels had shorter overall survival than those with higher levels, although not significantly so ( P = 0.084). In conclusion, 5-hmC expression was decreased in ESCC tissues, and was associated with TET2 expression level. TET2 reduction and subsequent 5-hmC loss might affect ESCC development.

The aim of this study was to evaluate the efficacy of the auditory evoked potential (AEP) index (aepEX) as an assessment tool for hypnosis during induction of various anesthetic drugs, and to compare its performance to that of the bispectral index (BIS). A total of 45 cases were divided into three groups based on the drugs used for anesthesia. Before anesthetic induction, BIS and AEP monitors were initiated. Anesthesia was induced through intravenous injection (IV) as follows: MP (n = 15) group, midazolam (0.1 mg/kg IV); TP (n = 15) group, thiopental (4 mg/kg IV); and KP (n = 15) group, ketamine (2 mg/kg IV). After loss of response (LOR), an infusion of 3 μg/ml propofol via a target-controlled infusion was initiated in all groups. AepEX and BIS were measured in the waking state (baseline) and at LOR (1 min after LOR), pre-intubation (1 min after previous intubation) and post-intubation (1 min after tracheal intubation finished). The value of aepEX significantly decreased in all groups with LOR and that of BIS also decreased except of KP group. No significant difference were observed in BIS values between baseline and LOR in the KS group. The aepEX might be more useful than BIS for hypnosis monitoring during anesthetic induction, particularly when drugs such as ketamine are used.

Titanium tetra-n-butoxide was hydrolyzed in the presence of benzoylacetone (BzAc), and the solution obtained was concentrated and served for spin-coating or dropping on substrates, followed by successive drying at 120, 200 and 250 °C. The dried products were transparent and amorphous, and the infrared absorption and Raman spectroscopic studies showed that BzAc forms chelate rings. Thermomechanical analysis showed that the 120 and 200 °C-dried products showed steep, thermoplastic shrinkage at around 30 and 70–85 °C, respectively, whereas the 250 °C-dried product did not show thermoplasticity. Thus as the drying temperature was increased, the thermoplasticity appeared at a higher temperature and finally disappeared. These changes in thermoplasticity with drying temperature were concluded to result from the progress of condensation between titanoxane polymers and/or clusters, which was evidenced in gel permeation chromatographic analysis.

The herpes simplex virus 1 (HSV-1) Us8A gene overlaps the gene that encodes glycoprotein E (gE). Previous studies have investigated the roles of Us8A in HSV-1 infection using null mutations in Us8A and gE; therefore, the role of Us8A remains to be elucidated. In this study, we investigated the function of Us8A and its phosphorylation at serine 61 (Ser-61), which we recently identified as a phosphorylation site by mass spectrometry-based phosphoproteomic analysis of HSV-1-infected cells, in HSV-1 pathogenesis. We observed that (i) the phosphorylation of Us8A Ser-61 in infected cells was dependent on the activity of the virus-encoded Us3 protein kinase; (ii) the Us8A null mutant virus exhibited a 10-fold increase in the 50% lethal dose for virulence in the central nervous system (CNS) of mice following intracranial infection compared with a repaired virus; (iii) replacement of Ser-61 with alanine (S61A) in Us8A had little effect on virulence in the CNS of mice following intracranial infection, whereas it significantly reduced the mortality of mice following ocular infection to levels similar to the Us8A null mutant virus; (iv) the Us8A S61A mutation also significantly reduced viral yields in mice following ocular infection, mainly in the trigeminal ganglia and brains; and (v) a phosphomimetic mutation at Us8A Ser-61 restored wild-type viral yields and virulence. Collectively, these results indicate that Us8A is a novel HSV-1 virulence factor and suggest that the Us3-mediated phosphorylation of Us8A Ser-61 regulates Us8A function for viral invasion into the CNS from peripheral sites. IMPORTANCE The DNA genomes of viruses within the subfamily Alphaherpesvirinae are divided into unique long (UL) and unique short (Us) regions. Us regions contain alphaherpesvirus-specific genes. Recently, high-throughput sequencing of ocular isolates of HSV-1 showed that Us8A was the most highly conserved of 13 herpes simplex virus 1 (HSV-1) genes mapped to the Us region, suggesting Us8A may have an important role in the HSV-1 life cycle. However, the specific role of Us8A in HSV-1 infection remains to be elucidated. Here, we show that Us8A is a virulence factor for HSV-1 infection in mice, and the function of Us8A for viral invasion into the central nervous system from peripheral sites is regulated by Us3-mediated phosphorylation of the protein at Ser-61. This is the first study to report the significance of Us8A and its regulation in HSV-1 infection.

Optically transparent, ca. 200–800 nm thick TiO2-hydroxypropylcellulose (HPC) hybrid thin films were prepared from Ti(OC3H 7 i )4–HPC–HCl–H2O–C3H 7 i OH solutions by the sol–gel method, where the as-deposited films were dried at 120 °C. The effects of the amount of HPC, H2O and HCl in the starting solutions on the refractive index and uncracking critical thickness of the films were studied, where the effects on the critical thickness was discussed on the basis of in situ stress measurements during heating. The increase in HPC content increased the critical thickness and lowered the refractive index. The increase in HCl content resulted in a decrease in critical thickness and an increase in refractive index. Larger H2O contents gave rise to a maximum in critical thickness while the refractive index was unaffected. Such variation in critical thickness with varying solution compositions was demonstrated to result from the differences in in-plane stress generated during heating. By optimizing the processing parameters an 810 nm thick TiO2–HPC hybrid film of a refractive index of 1.84 was obtained.

Prostaglandin E(1) (PGE(1)) has been shown to provide short-term neuroprotection against various types of brain ischemia in a dose-dependent manner in mice. However, these findings were obtained from experiments performed without any control over physiological parameters. We performed an outcome study where physiological parameters were controlled in an attempt to confirm the dose-dependant neuroprotective effects of PGE(1).A rat model of severe forebrain ischemia was used. Two doses of PGE(1) were administered during the pre-ischemic period, a low dose (LowPG group) and a high dose (HighPG group). Normotension was maintained in the LowPG group, while hypotension was induced in the HighPG group. In separate groups, normal saline (Control) or sodium nitroprusside (SNP) were infused to compare outcomes under similar blood pressure conditions. Histological outcomes in the hippocampal CA1 and entorhinal cortex were evaluated 5 days post-ischemia.HighPG resulted in hyperglycemia. The percentage of dead neurons in the hippocampal CA1 and entorhinal cortex were similar in the Control, SNP, and HighPG groups, the percentage being significantly attenuated in the LowPG group (CA1: Control = 92.8 +/- 2.4%, LowPG = 85.0 +/- 8.5%, HighPG = 95.3 +/- 2.4%, and SNP = 96.4 +/- 0.7%, P0.01; entorhinal cortex: Control = 73.8 +/- 4.0%, LowPG = 53.2 +/- 12.3%, HighPG = 72.1 +/- 12.6%, and SNP = 76.5 +/- 4.1%, P0.01).Pre-ischemic administration of low-dose PGE(1) in rats provided neuroprotection against severe forebrain ischemia. A dose dependency was not observed with PGE(1) dose and outcome.

DNA polymerase lambda, pol lambda, is a eukaryotic member of the X-family DNA polymerases that is involved in two modes of DNA repair, i.e. base excision repair (BER) or non-homologous end joining (NHEJ). Using immunohistochemical approaches, we have observed pol lambda expression in human tissues, particularly in the respiratory system of lung cancer patients. pol lambda proteins were distributed in the nuclei of the epithelial cells in the bronchi, bronchioles and alveoli. Intriguingly, the level of pol lambda expression in the bronchiolar epithelia significantly correlated with the amount of habitual smoking in the individuals. Conversely, pol lambda expression in cancer tissues did not correlate with the smoking status of the patients. Pol lambda expression was sometimes discrepant between the tumor tissues and adjacent bronchioles. More importantly, tumors without pol lambda expression that occurred in heavy smokers significantly tended to be at an advanced clinical stage. Pol lambda may thus be involved in the DNA repair processes counteracting DNA damage caused by tobacco smoke in the respiratory system.

(NaNbO3)1-x(BaTiO3)x (NN-xBT) thin films with low BaTiO3 (BT) concentrations x (x = 0.05 and 0.10) were fabricated on SrRuO3/(001)SrTiO3 (SRO/(001)STO) substrate by pulsed laser deposition (PLD). X-ray diffraction pattern (XRD) and transmission electron diffraction pattern (TED) showed that NN-0.10BT thin film was epitaxially grown on SRO/(001)STO substrate with a crystallographic relationship of [001]NN-xBT||[001]STO. From reciprocal space maps, the lattice parameters of the out-of-plane direction of NN-xBT thin films became larger with an increase in BT concentration, although the lattice parameter of the in-plane was hardly changed by the BT concentration. The value of relative dielectric constant er of the NN-xBT thin films were increased with BT concentration. The er and the dielectric loss tanδ of NN-0.10BT were 1220 and 0.02 at 1 kHz, respectively. The P-E hysteresis loops of the NN-xBT thin films showed clear ferroelectricity. Although the value of remanent polarization Pr decreased with the BT concentration, the behaviors of er, Pr, and coercive electric field Ec of the NN-xBT thin films against the BT concentration accorded with those of NN-xBT ceramics, in which NN-0.10BT ceramics exhibited the largest piezoelectric property. Therefore, the NN-0.10BT thin film is expected to show high piezoelectricity.

The purpose of this study was to clarify the effect of ethanol on rat molar tooth development. Pregnant rats were fed 25%-ethanol solutions or ethanol-free water up to delivery, and tooth germs, as well as fully developed teeth, obtained from the offspring (ethanol-exposed rats or control rats) were subjected to morphometric analysis and immunohistochemistry. No significant differences were observed in terms of tooth size between the ethanol-exposed and control rats. Moreover, no abnormalities in cusp number, form or dental surface structure was noted in the ethanol-exposed rats. In the ethanol-exposed rats, the tooth development showed a slight retardation in comparison to the controls. Morphological alteration in tooth germs was not observed in the experimental rats. Both bone morphogenetic protein-4 and basic fibroblast growth factor-2 were immunolocalized in the cells composing the tooth germs in the ethanol-exposed rats. No differences, however, were found in immunostaining intensity between the ethanol-exposed and control rats. Taken together, the results indicate that the effect of ethanol on tooth development was weaker than expected in the rat FAS model.

Aims: Nitric oxide (NO), produced by inducible NO synthase (iNOS), has been suggested to cause oxidative stress, leading to 8-hydroxydeoxyguanosine (8-OHdG) accumulation and subsequent transversion mutation of DNA. The aim was to evaluate iNOS expression and the status of oxidative stress in nasopharyngeal carcinoma (NPC). Methods and results: Seventy-three cases of NPC were investigated to examine the immunohistochemical expression of iNOS, 8-OHdG and latent membrane protein-1 (LMP-1) and Epstein–Barr virus-encoded small RNA (EBER) expression using in situ hybridization. iNOS mRNA expression and p53 gene mutations were also assessed. Overexpression of iNOS, LMP-1 and EBER was observed in 62 (84.9%), 28 (38.4%) and 53 (72.6%) cases respectively. p53 gene mutation was found in 10 of 73 (13.7%) cases. Immunohistochemical iNOS expression was associated with the 8-OHdG labelling index, iNOS mRNA expression and p53 gene alteration (P

Organically modified polyoxotitanates were prepared by hydrolyzing titanium tetra-n-butoxide in the presence of benzoylacetone (BzAc) in solution at Ti(OC4H9)4 : BzAc : H2O : CH3COCH3 mole ratios of 1 : (1 or 2) : (1 or 10) : 20. The solutions were concentrated at 80°C and dried at 140°C in N2 flow, producing transparent and yellow materials of BzAc-modified polyoxotitanate. The materials had refractive indices of ∼1.7 and exhibited thermoplastic softening at temperatures below 70°C. Low contents of BzAc and high contents of H2O in solution led to higher softening temperatures of the materials, indicating the tunability of the softening temperature. Gel permeation chromatographic analyses showed that the higher softening temperatures originated mainly from polymerized species of higher molecular weights that were generated by hydrolysis and condensation reactions promoted by the low BzAc and high H2O contents in solution. © 2015 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2015, 132, 42653.

Replacement of the herpes simplex virus 1 small capsid protein VP26 phosphorylation site Thr-111 with alanine reduced viral replication and neurovirulence to levels observed with the VP26 null mutation. This mutation reduced VP26 expression and mislocalized VP26 and its binding partner, the major capsid protein VP5, in the nucleus. VP5 mislocalization was also observed with the VP26 null mutation. Thus, we postulate that phosphorylation of VP26 at Thr-111 regulates VP26 function in vitro and in vivo .

Malignant peripheral nerve sheath tumor (MPNST) is an uncommon soft tissue neoplasm with a poor prognosis, occurring sporadically or associated with neurofibromatosis type 1 (NF1); however, the histogenesis of MPNST remains unclear, especially in sporadic tumors. There are two major forms of genomic instability in human cancer: chromosomal instability (CIN) and microsatellite instability (MSI). An inverse relationship has recently been demonstrated between CIN and MSI in colorectal cancers. CIN and MSI are suggested to be individual pathways, which are involved in the pathogenesis and which may lead to specific clinical and pathological characteristics. To elucidate the chromosomal aberration as a consequence of CIN and MSI status of MPNST, we karyotyped 10 MPNSTs from nine patients, and examined the MSI of seven microsatellite markers using high-resolution fluorescence microsatellite analysis; 2 out of 10 cases (20%) had normal karyotypes, and 8 out of 10 cases (80%) revealed structural and numerical chromosomal aberrations. Three of the 10 cases (30%) showed near triploidy. The most frequent aberration was -22 (40%), followed by +2, +14, -13, -17, and -18 (30% each). An MSI-low status was observed in 30% of cases; the remaining cases showed microsatellite stability. These findings suggest that chromosomal aberration as a consequence of CIN has a greater role in the pathogenesis of MPNST than does that due to MSI.

Background Hypoxia-inducible factor 1α (HIF-1α) plays a key role in responses to hypoxia and expression of HIF-1α downstream genes leads to both an adapted metabolism and increased oxygen supply. We investigated the clinical significance of HIF-1α expression in gastric carcinoma. Methods We examined HIF-1α, vascular endothelial growth factor (VEGF), and insulin-like growth factor-2 (IGF-2) expression patterns immunohistochemically in 126 specimens of gastric carcinoma. CD34 antigen levels were also examined by immunohistochemistry to determine microvessel density (MVD) within tumors and correlations between HIF-1α expression, clinicopathological features, and survival were examined. Results HIF-1α expression correlated with tumor size (P

ADRP is associated with intracellular lipid droplets. We demonstrate the regulatory mechanism for ADRP expression in RAW264.7 macrophages. The ADRP mRNA expression was stimulated by PMA, and synergistically enhanced in association with its protein level in the presence of lipids. A proteasome inhibitor protected the protein from degradation under the lipid-free conditions. One of the possible sites of the PMA action was proved to be an Ets/AP-1 element in the promoter, since mutations of this site reduced the PMA-induced promoter activity, and ligation of this element led to a significant increase in the PMA-responsiveness of homologous or heterologous promoters. Mutations of this site diminished the synergistic effect on the promoter activity induced by PMA and oleic acid, suggesting a possible interaction between this site and the downstream PPARdelta site. EMSA revealed that PU.1 and AP-1 conjointly bound to this site. The juxtaposition of the two sequences was requisite for full activity, since spacer sequences between them decreased the PMA-induced activity. PI3 kinase inhibitor was found to reduce the PMA-induced mRNA expression and promoter activity in parallel with PU.1/AP-1 complex formation on EMSA. From these results, we concluded that the Ets/AP-1 site is an important cis-acting element that regulates the ADRP gene expression in macrophages.

Microsatellite instability (MSI) is associated with defective DNA mismatch repair in various human malignancies. Using a unique fluorescent technique, we have observed two distinct modes of dinucleotide microsatellite alterations in human colorectal cancer. Type A alterations are defined as length changes ofor =6 bp. Type B changes are more drastic and involve modifications ofor =8 bp. We show here that defective mismatch repair is necessary and sufficient for Type A changes. These changes were observed in cell lines and in tumours from mismatch repair gene-knockout mice. No Type B instability was seen in these cells or tumours. In a panel of human colorectal tumours, both Type A MSI and Type B instability were observed. Both types of MSI were associated with hMSH2 or hMLH1 mismatch repair gene alterations. Intriguingly, p53 mutations, which are generally regarded as uncommon in human tumours of the MSI+ phenotype, were frequently associated with Type A instability, whereas none was found in tumours with Type B instability, reflecting the prevailing viewpoint. Inspection of published data reveals that the microsatellite instability that has been observed in various malignancies, including those associated with Hereditary Non-Polyposis Colorectal Cancer (HNPCC), is predominantly Type B. Our findings indicate that Type B instability is not a simple reflection of a repair defect. We suggest that there are at least two qualitatively distinct modes of dinucleotide MSI in human colorectal cancer, and that different molecular mechanisms may underlie these modes of MSI. The relationship between MSI and defective mismatch repair may be more complex than hitherto suspected.

Summary Background : In non-small cell lung cancer, a loss of heterozygosity (LOH) is frequently observed; however, few studies have investigated the differences in the LOH status between adenocarcinoma and squamous cell carcinoma. Patients and methods : In a consecutive series of 49 patients with adenocarcinomas and 22 patients with squamous cell carcinomas, the LOH in tumors was analyzed using polymerase chain reaction employing 5 fluorescence-labeled dinucleotide markers (D2S123, D5S107, D10S197, D11SS904, D13S175) and an autosequencer. Results : LOH was more frequently observed in squamous cell carcinoma (20 of 22, 90%) than in adenocarcinomas (33 of 49, 67%) ( P = 0.0348 ), and the number of LOH per patient was also higher in the patients with squamous cell carcinoma (2.2±1.4) than in those with adenocarcinoma (1.5±1.2, P = 0.037 ). In adenocarcinomas, the number of LOH per patients correlated significantly with the pack-year index, whereas the pathological stage significantly affected the number of LOH in squamous cell carcinomas. Conclusion : The presence of LOH is relatively uncommon in adenocarcinoma of the lung; however, the incidence of LOH tends to be associated with the smoking status.

Synchronous tumors of the ovary and endometrium are a well-known phenomenon. There are histological criteria for defining double primary tumors or metastasis from one organ to another, but in some cases a precise diagnosis is difficult. In this study we reviewed 17 cases of synchronous ovarian and endometrial adenocarcinoma by previously reported histological criteria and performed a microsatellite analysis, combined with X-linked clonality analysis. We also analyzed 8 cases of endometrial adenocarcinoma with pelvic lymph node metastasis as a control. Five dinucleotide microsatellite markers were selected, and microsatellite analysis was performed by a high-resolution method using fluorescence-labeled polymerase chain reaction and laser scanning. In synchronous tumors, 11 ovarian carcinomas (65%) and 10 endometrial carcinomas (59%)demonstrated microsatellite instability (MSI). In total, 13 of the 17 patients demonstrated MSI in the ovarian tumor, the endometrial tumor, or both. Four cases of endometrial carcinoma with pelvic lymph nodes metastases displayed MSI, and MSI findings of the endometrial tumor and lymph node metastasis were same in all cases. Based on these findings, we considered that similar MSI findings indicate metastatic tumors. According to the MSI findings, 13 of the 17 patients (76%) had single or double clonal tumors, 11 (67%) with double primary tumors and 2 (13%) with metastatic tumors. Using X-linked clonality analysis, 3 patients were diagnosed with double primary tumors. The molecular diagnosis corresponded with the histological criteria in all but 1 case. In conclusion, using both MSI and X-linked clonality analysis, most patients (82%) could be diagnosed as having single or double clonal tumors. The histological criteria are accurate and useful in most cases; however, in some cases where the relationship between the 2 tumors is difficult to determine, high-resolution MSI analysis may be helpful.

Dermatofibrosarcoma protuberans (DFSP) is a dermal and subcutaneous tumor categorized as a tumor of intermediate malignancy, and its progression in some cases to fibrosarcoma is well known. However, molecular analysis of tumor progression has been limited. The present study investigated microsatellite instability (MSI) of 7 microsatellite markers through high-resolution microsatellite analysis in addition to a mutational analysis of the p53 gene in 44 tumors in 36 patients. The patients were divided into 2 groups: 9 patients with a fibrosarcomatous component in the primary or recurrent/metastasized tumor, designated as the DFSP+FS group, and the remaining 27 patients, designated as the DFSP group. Cases in which the percentage of markers with an additional peak among the markers successfully analyzed was more than 30% was considered MSI high (MSI-H); cases in which microsatellites were stable at all of the successfully examined markers were considered microsatellite stable (MSS); and the remaining cases were considered MSI low (MSI-L). MSI-H cases were observed more frequently in the DFSP+FS group (4 of 9 cases) than in the DFSP group (1 of 27 cases) (P = 0.028, Fischer's exact test). The MSI status of recurrent or metastatic tumors in both the DFSP+FS and the DFSP groups was the same as that in the corresponding primary neoplasms. Furthermore, there was no difference in MSI status between an ordinary DFSP area and a fibrosarcomatous area in 7 tumors that exhibited both areas. p53 mutational analysis revealed 10 point mutations, composed of 4 missense mutations and 6 silent mutations, in 6 of 36 cases (16.7%). A missense mutation was more frequently observed in the DFSP+FS group (3 of 4) than in the DFSP group (1 of 4). Among 3 cases of a missense mutation in the DFSP+FS group, 2 had a mutation only in a fibrosarcomatous area and 1 had a mutation only in a metastatic tumor progressing to fibrosarcoma. These results suggest that MSI and p53 mutations are involved in tumor progression of DFSP to fibrosarcoma as early and late events, respectively.

Background: We recently developed a novel system for detecting microsatellite alteration, which is an important process in carcinogenesis. In patients with non-small cell lung cancer (NSCLC), loss of heterozygosity (LOH) is frequently observed and causes functional disorders of tumor suppressor genes. Patients and methods: In a consecutive series of 51 patients with NSCLC who had undergone a surgical resection, microsatellite instability (MSI) and LOH in tumors were analyzed by polymerase chain reaction using five fluorescence-labeled dinucleotide markers (D2S123, D5S107, D10S197, D11SS904, and D13S175) and an autosequencer. Results: MSI was detected in only one patient (2.0%) with only one marker. LOH was detected in at least one chromosomal region that was tested in 39 patients (76%). The mean ( SD) number of LOHs detected by each marker was 1.74 1.40, with 1 LOH detected in 10 patients, 2 LOHs detected in 15 patients, 10 LOHs detected in 3 patients, 1 LOH detected in 4 patients, and 3 LOHs detected in 5 patients. The number of LOHs detected in each patient was significantly associated with the pack-year index ( 0.501; p 0.0004), although there was no relationship with having a history of multiple cancers and familial cancer. Patients with stage IA disease showed a significantly lower number of LOHs than did patients with other stages of disease (1.15 vs 2.38, respectively; p 0.0013). Conclusion: LOH is very common in patients with NSCLC, and the number of LOHs increases with increases in smoking, suggesting the presence of an important event in lung carcinogenesis. (CHEST 2003; 123:545–550)

Mismatched or damaged base pairs in DNA are mutagenic and both eukaryotes and prokaryotes have a series of repair systems that decrease a spontaneous mutation rate. All exocyclic amino groups of cytosine(C), adenine(A), and guanine(G) contribute to hydrogen bonds for base pairing. High temperature and oxidative stresses increase the deamination of these bases and methylated C. These deaminated sites would be initially recognized by components of DNA repair system. We discovered a novel G/thymine(T)-mismatch binding protein (nGTBP) that bound, with high affinity, to a minimal 14-mer DNA heteroduplex with a strict 5'-TRT GNB-3' sequence (R for purine, N for any bases, and B for "not A," namely for C, G, or T ). This italicized G position mismatched with T could be replaced by hypoxanthine, the deaminated A. The nGTBP, however, barely recognized DNA duplexes individually containing 8-oxo-G, thymine glycol, and 5-methylcytosine.

We prepared a new class of organic–inorganic hybrid materials with high refractive indices and thermoplasticity. Titanium and zirconium alkoxides were hydrolyzed in the presence of β-diketones of h...

Background: Various studies have analyzed the relationships between new biological parameters and DNA ploidy for human cancer. However, the biological significance of DNA ploidy remains unclear, since there have so far been few studies analyzing DNA ploidy in relation to the combination of multiple such biological parameters. Methods: Samples for the analysis of DNA ploidy, estrogen receptor (ER), progesterone receptor (PgR), p53 protein and epidermal growth factor receptor (EGFR) were prepared from the same frozen specimens of 498 primary breast cancers. DNA ploidy was determined by flow cytometry, while ER and PgR were determined by an enzyme immunoassay. Both p53 protein and EGFR were determined by immunohistochemical analyses. Results: Aneuploidy was significantly correlated with the absence of ER, the absence of PgR, the positivity of p53 protein and the positivity of EGFR, and it was also significantly correlated with the combination of the absence of ER and PgR and the combination of the positivity of p53 protein and EGFR. A deviation in the four parameters was defined as the absence of ER, the absence of PgR, the positivity of p53 protein or the positivity of EGFR. As the number of deviations of these four biological parameters increased, the incidence of aneuploidy increased significantly (p < 0.0001). Multivariate analysis also indicated that deviation of two, three or all four parameters were significant factors for DNA ploidy, while the p value decreased and the odds ratio increased as the number of deviations of the four biological parameters increased. Conclusions: DNA aneuploidy based on flow cytometric analysis reflects the accumulation of deviations in the four biological parameters investigated in the present study. The two facts that DNA aneuploidy reflects the accumulation of the aggressiveness of these biological parameters and that DNA aneuploidy consisted of heterogeneous characteristics which were represented by the deviation of each biological parameter should be taken into consideration when selecting treatment strategies for breast cancer.

Yersinia enterocolitica was isolated from 15.7% (88/560) of wild rodents captured in 15 prefectures in Japan. Prevalences by rodent species were 18.0% (70/388) in Japanese field mice (Apodemus speciosus), 20% (14/71) in small Japanese field mice (Apodemus argenteus), and 11% (4/38) in gray red-backed vole (Myodes rufocanus bedfordiae), suggesting that these rodent species are important reservoirs of Y. enterocolitica. Although most of the isolates were identified as biotype 1A, the pathogenic bioserotype 1B/O:8 was detected in one of the A. speciosus and in three of the A. argenteus captured in Aomori Prefecture. It is suggested that Apodemus mice may be an important reservoir of Y. enterocolitica, and that there are foci of the pathogenic bioserotype 1B/O:8 in Aomori Prefecture, because human sporadic cases by the serotype have been reported in this prefecture.

We experienced severe asthmatic crisis during general anesthesia in a 45-year-old man with IgG4-related disease, COPD and athma undergoing removal of submandibular gland. The ventilatiory failure was caused by the stimulation of the operation, sputum, and neostigmine. His serum IgG4 level was extremely high. IgG4 related disease is a recently emerging entity characterized by a diffuse or mass forming inflammatory reaction rich in IgG4-positive plasma cells associated with fibrosclerosis and obliterative phlebitis. It is associated with an elevated serum level of IgG4 and an allergic disease. We must be careful in perioperative management of the patients with IgG4-related disease because general anesthesia can induce asthmatic crisis.